Roles of lipocalin-type and hematopoietic prostaglandin D synthases in mouse retinal angiogenesis

Normal angiogenesis is essential for retinal development and maintenance of visual function in the eye, and its abnormality can cause retinopathy and other eye diseases. Prostaglandin D2 is an anti-angiogenic lipid mediator produced by lipocalin-type PGD synthase (L-PGDS) or hematopoietic PGD synthase (H-PGDS). However, the exact role of these PGD synthases remains unclear. Therefore, we compared the roles of these synthases in murine retinal angiogenesis under physiological and pathological conditions. On postnatal day (P) 8, the WT murine retina was covered with an elongated vessel. L-PGDS deficiency, but not H-PGDS, reduced the physiological vessel elongation with sprouts increase. L-PGDS expression was observed in endothelial cells and neural cells. In vitro, L-PGDS inhibition increased the hypoxia-induced vascular endothelial growth factor expression in isolated endothelial cells, inhibited by a prostaglandin D2 metabolite, 15-deoxy-Δ12,14 -PGJ2 (15d-PGJ2) treatment. Pericyte depletion, using antiplatelet-derived growth factor receptor-β antibody, caused retinal hemorrhage with vessel elongation impairment and macrophage infiltration in the WT P8 retina. H-PGDS deficiency promoted hemorrhage but inhibited the impairment of vessel elongation, while L-PGDS did not. In the pericyte-depleted WT retina, H-PGDS was expressed in the infiltrated macrophages. Deficiency of the D prostanoid receptor also inhibited the vessel elongation impairment. These results suggest the endogenous role of L-PGDS signaling in physiological angiogenesis and that of H-PGDS/D prostanoid 1 signaling in pathological angiogenesis.

Angiogenesis is a process in which new blood vessels branch from pre-existing vessels and is crucial for maintaining homeostasis under physiological and pathological conditions (1).Physiological angiogenesis is observed in tissue development and repair, while pathological angiogenesis is observed in chronic inflammation.In the eye, normal angiogenesis is also indispensable for retinal development and maintenance of visual function, of which abnormality can lead to some eye diseases, including retinopathy and macular degeneration.
PGD 2 is one of the major PGs synthesized by lipocalintype PGD synthase (L-PGDS) and hematopoietic PGD synthase (H-PGDS).L-PGDS is expressed in the central nervous system, by astrocytes and oligodendrocytes, under the regulation of NF-E2-related factor 2 and activator protein (AP)-1 (10,11).H-PGDS is expressed in inflammatory cells, such as mast cells, megakaryocytes, and macrophages under the regulation of Ras guanyl nucleotide-releasing protein 4, organic cation transporter-1, and AP-2 (12,13).The synthesized PGD 2 works as a ligand of two distinct G protein-coupled receptors: D prostanoid (DP) 1 and DP2.Our group previously showed that gene deficiency of both L-PGDS and H-PGDS increased vascular permeability and angiogenesis in murine lung carcinoma models (14,15).Although these results highlight the anti-angiogenic effect of PGD 2 signaling in tumor growth, the difference and/or significance of these synthases in angiogenesis remain unclear.
In the present study, we aimed to compare the role of two PGD synthases, L-PGDS and H-PGDS, in angiogenesis by focusing on two murine retinal angiogenesis: neonatal physiological angiogenesis and PC depletioninduced pathological angiogenesis.
P1 neonatal mice were administrated with rat anti-PDGFRβ antibody (APB5, intraperitoneally, 50 μg/50 μl in PBS; purified as previously described ( 6)).The control mice were administrated with only PBS.After the administration, mice were euthanized by cervical dislocation, and the eyes were excised and analyzed for further experiments.

Morphological analysis of mice retina
Excised mice eyes of postnatal day (P) 4, P8, and P14 were fixed with 4% paraformaldehyde (PFA) in PBS for 30 min.After the fixation, retinal cups were dissected and fixed again with 4% PFA overnight.
The vessel elongation was calculated by averaging the distances from the optic nerve to the end of the angiogenic front in each slice.The number of sprouts was also calculated by averaging the counted sprouts number in the angiogenic front of each slice.Finally, the number of macrophages was calculated from the averaged counts in four 300 × 300 μm fields in the angiogenic area.

Real-time PCR of mice retina
Retinal cups were excised from the eyes of P4 and P8 mice.Total RNA was extracted using TRI-Reagent (Molecular Research Center, OH).The complementary DNA (cDNA) was obtained by reverse transcription using a Random 9-mer primer (TOYOBO, Tokyo, Japan) and ReverTra Ace (TOYOBO) at 30 • C for 10 min, 42 • C for 1 h, and 99 • C for 5 min according to the manufacturer's protocol.Then, the cDNA was amplified for 45 cycles of 95 • C for 15 s and 59 • C for 1 min, using Platinum SYBR Green qPCR SuperMix-UDG (Thermo Fisher Scientific, MA) and AriaMx Real-Time PCR System (Agilent Technologies, CA).The mRNA expression levels were quantitated with the ΔΔCt method, using 18S-rRNA levels as internal controls.The list of primer sequences is shown in supplemental Table S1.

Scoring retina
We scored excised APB5-treated retinal cups as described previously (8), which were defined as follows: grade 0, retina with no hemorrhage or edema; grade 1, local hemorrhage; grade 2, hemorrhage and/or mild edema; grade 3, edema in up to one-half of the retina; and grade 4, the collapse of the retina.
The total RNA of HUVECs was extracted, and cDNA was obtained by reverse transcription.The cDNA was amplified by using specific primers (supplemental Table S2).The mRNA expression levels were quantitated with the ΔΔCt method, using 18S-rRNA levels as internal controls.
We measured the level of PGE 2 (one of major PGs as a control) and PGD 2 in P4 and P8 retina using LC/MS system.L-PGDS or H-PGDS deficiency did not affect the level of PGE 2 in P4 and P8 retina (Fig. 1C).On the other hand, both L-PGDS and H-PGDS deficiency significantly decreased the level of retinal PGD 2 in P4 mice, not in P8 (Fig. 1D).Although we also tried to measure the retinal level of Δ12-PGJ 2 and 15d-PGJ 2 , we could not detect them in P4 and P8 retina (data not shown).
Next, we investigated the angiogenesis front of the retinal vessels.As shown in Fig. 1E, F, L-PGDS deficiency significantly increased the number of sprouts on both P4 and P8.We also assessed the diameter of the retinal vein and artery near the optic papilla as indices of vessel abnormalities (supplemental Fig. S1C).However, these parameters did not differ between WT, L-pgds −/− , or H-pgds −/− mice (supplemental Fig. S1D).
These results suggest that L-PGDS, but not H-PGDS, was required for vessel elongation and sprouts redocumentation in the neonatal retinal angiogenesis.

Endothelial and neural L-PGDS reduced the expressions of pro-angiogenic factors
To clarify the L-PGDS-producing cell, immunofluorescent staining was performed in the cross sections of the P8 WT retina (Fig. 2A).L-PGDS was localized in isolectin B4-positive ECs (Fig. 2B, upper panels) and PGP9.5-positive neural cells (lower panels).The retina is divided into several neuron cell layers.L-PGDS expression was observed in the ganglion cells and inner nuclear cell layers.In addition, we confirmed the existence of L-PGDS (L-pgds) mRNA in the P4 and P8 retinas, which were suppressed by L-PGDS deficiency (Fig. 2C).In addition, we also measured the mRNA expression of PGD 2 -related genes: H-PGDS, DP1, and DP2.L-PGDS deficiency significantly increased the mRNA level of H-PGDS in P4 retina, not in P8 retina (H-pgds, Fig. 2D).On the other hand, L-PGDS deficiency did not affect the mRNA expression of DP1 (Dp1, Fig. 2E) and DP2 (Dp2, Fig. 2F).
We further investigated the role of L-PGDS in the mRNA levels of vascular growth factors.L-PGDS deficiency significantly increased the mRNA level of VEGF-A (Vegfa, Fig. 2G) but not FGF-2 (Fgf2, Fig. 2H), EGF (Fgf, Fig. 2I), or VEGFR2 (Kdr, Fig. 2J) in the P4 retina.In the P8 retina, L-PGDS deficiency significantly increased the mRNA levels of FGF-2 and EGF but not VEGF-A or VEGFR2.
These results suggest that L-PGDS signaling reduced excessive expression of pro-angiogenic factors, which probably leads to chaotic and dysregulated vessel elongation at P8.

H-PGDS deficiency aggravated symptoms in PC depletion-induced retinopathy model
We then investigated the roles of L-PGDS and H-PGDS in pathological retinal angiogenesis using PC depletion-induced retinopathy model.The administration of an anti-PDGFRβ antibody, APB5, depletes PCs in developing mouse retinas.This PC depletion leads to clinical symptoms of human diabetic retinopathy, including blood-retina barrier breakdown, retinal edema, and hemorrhage (6).In the vehicle-treated P8 WT retina, retinal isolectin B4-positive ECs were covered by desmin-positive PCs (supplemental Fig. S2A, left panel).The administration of APB5 (50 μg in 50 μl PBS/head at P1, intraperitoneally) depleted PCs around ECs in P8 WT, L-pgds −/− , and H-pgds −/− mice (supplemental Fig. S2A; right panels).The administration of APB5 also caused bleeding (black arrowheads) inside the retinal cups in P8 WT mice (Fig. 4A).L-PGDS deficiency did not differ from the bleeding in WT, while H-PGDS deficiency seemed to be aggravated the bleeding in APB5-treated retinas.We also quantified "retinal grading score" that represents the level of The role of prostaglandin D synthases in angiogenesis retinal edema and hemorrhage.PC depletion significantly increased the score in P8 WT (Fig. 4B), which H-PGDS deficiency but not L-PGDS significantly increased.
The APB5-induced PC depletion is also known to causes distortion, shortness, and dilation of retinal vessels (6).These are characteristic remark of pathological angiogenesis.PC depletion in WT mice induced the decrease of vascular elongation accompanied by impaired vascular structure (Fig. 4C, D; control WT, 2.0 ± 0.1 mm; PC-depleted WT, 1.6 ± 0.1 mm).Contrary to the results of the retinal grading score, H-PGDS deficiency inhibited the APB5-induced shortage of vascular elongation (PC-depleted H-pgds −/− mice, 1.9 ± 0.1 mm).L-PGDS deficiency did not affect the vessel elongation (PC-depleted L-pgds −/− mice, 1.6 ± 0.1 mm).In addition, in WT mice, APB5 administration tended to increase the diameter of the vein, but not the artery (supplemental Fig. S2B, C).Neither L-PGDS nor H-PGDS deficiency affected the diameter of the veins or arteries.
These results suggest that H-PGDS, but not L-PGDS, is involved in PC depletion-induced pathological angiogenesis.

H-PGDS is located in the macrophage of PC-depleted P8 retina
We next investigated the H-PGDS-expressing cell using en-face immunofluorescence staining.In PC-depleted WT retinas, H-PGDS expression was observed in CD68-positive macrophages (Fig. 5A, upper panels, white arrows).We confirmed that H-PGDS expression was not observed in H-PGDS-deficient retina (lower panels).
Several studies have shown that PC depletion causes inflammation indicated as vascular hyperpermeability and macrophage infiltration (6,8).We tried to clarify the effect of H-PGDS on the PC depletion-induced inflammatory responses.In control P8 WT retina, en-face immunostaining showed that fibrinogen (plasma protein, green) was observed inside the retinal vessel (Fig. 5B, left panel, white arrowhead).PC depletion caused fibrinogen extravasation, representing vascular hyperpermeability (right panels).Compared to WT, fibrinogen extravasation seemed to be stronger in H-pgds −/− retinas but not in L-pgds −/− .In WT retinas, PC depletion also caused F4/80-positive macrophage infiltration (supplemental Fig. S3A, B, left panel).However, H-PGDS or L-PGDS deficiency did not affect the number of macrophage (right panels).
These results indicate that H-PGDS is expressed in macrophage, which inhibited vessel elongation without affecting macrophage infiltration.
This result suggests that DP1 was probably the downstream signaling of H-PGDS in the PC depletioninduced pathological angiogenesis.

DISCUSSION
In the present study, we investigated the role of L-PGDS and H-PGDS in physiological and pathological angiogenesis using mouse retina models.We detected L-PGDS expression in ECs and neural cells of the neonatal retina, while H-PGDS was expressed in macrophages in PC-depleted retina.In neonatal retinal angiogenesis model, L-PGDS deficiency, but not H-PGDS, DP1, or DP2 deficiency, reduced vessel elongation with the excessive transcription of pro-angiogenic factors.While in PC depletion-induced retinopathy model, H-PGDS and DP1 deficiency, but not L-PGDS or DP2, inhibited the APB5induced vessel elongation decrease but aggravated retinal grading score.
It is known that pro-angiogenic factors such as VEGF, FGF-2, and EGF play a crucial role in retinal angiogenesis.In murine neonatal retinal angiogenesis, tip cells at the angiogenic front ECs migrate according to the VEGF-A gradient from astrocyte (21).The costimulation with VEGF-A and FGF-2 on Matrigel plug-in mice abdomen synergistically promotes angiogenesis (22).Conversely, the excessive secretion of pro-angiogenic factors and/or excessive sprout formation impairs normal angiogenesis.Overexpression of VEGF induced chaotic sprout formation, resulting in delayed vessel elongation (23).Kim et al. also showed that deficiencies of large tumor suppressor half increased tip cell number, leading to decreased vessel elongation and dysregulated angiogenesis (24).Thus, regulating adequate proangiogenic factors is essential for neonatal retinal angiogenesis.In this study, we found that L-PGDS deficiency, but not H-PGDS deficiency, impaired normal retinal angiogenesis.L-PGDS deficiency also increased the number of sprouts with partial VEGF, FGF-2, and EGF expression increase.Consistent with previous studies (25,26), we confirmed that ECs and neural cells expressed L-PGDS in the P8 retina.Several inflammatory stimuli, such as cytokines and chemical substances, are known to upregulate L-PGDS expression in ECs (14,27).Notably, the hypoxia niche accelerates L-PGDSderived PGD 2 production in rat atria by activating hypoxia-inducible factor 1α (28).According to a study showing that the neonatal retina is hypoxic until P8 and changes to normoxic conditions by P14 (29).We can thus conclude that L-PGDS is a regulator for producing appropriate pro-angiogenic cytokines to maintain normal angiogenesis under hypoxic conditions.Unexpectedly, vessel formation was normalized on P14, even under L-PGDS deficiency.Several compensatory systems normalize excessive angiogenesis.Angiopoietin 1 (Ang 1) and its receptor (TIE2) signaling stabilize ECs by suppressing the transcription of angiogenic factors (30).Unc-5 Netrin Receptor B (UNC5B) and Roundabout Guidance Receptor 4 (Robo4) signaling also stabilizes angiogenesis by inhibiting VEGF signaling in the murine retina (31).These systems might reverse L-PGDSmediated inhibition of abnormal angiogenesis under normoxic conditions.
Unlike L-PGDS, H-PGDS deficiency did not affect physiological angiogenesis but did affect pathological angiogenesis.H-PGDS is expressed in hematopoietic cells, such as mast cells and macrophages.We found that H-PGDS was expressed in the infiltrated The role of prostaglandin D synthases in angiogenesis macrophages but not in the constituent cells, such as ECs and neural cells.The inducible expression of H-PGDS limits its functional role in physiological retinal angiogenesis.
PC depletion-induced retinopathy model produces clinical features of diabetic retinopathy, such as vascular tortuosity and dilation, edema, and hemorrhage (6).We found that H-PGDS-deficient retina, but not L-PGDS, increased vessel elongation compared with APB5-induced WT retina.H-PGDS deficiency also promoted retinal inflammation, especially hemorrhage and edema formation, as is shown from the results of retinal grading score.Our previous study showed similar results that, in the tumor implantation model, H-PGDS deficiency promotes vascular hyperpermeability and subsequent angiogenesis in implanted carcinomas (35).In the LPS-induced acute lung injury model and complete Freund's adjuvant-induced joint inflammation model, H-PGDS deficiency increased the levels of inflammatory cytokines, including TNFα, IL-1β, and SDF1α (37,38).Considering the H-PGDS expression in CD68-positive macrophages, the macrophages-derived H-PGDS might reduce PC depletion-induced retinal inflammation by decreasing vascular permeability or inflammatory cytokines.This signaling enhancement can be a new therapeutic tool for diabetic retinopathy.
We also found that DP1 deficiency, but not DP2, inhibited APB5-induced decrease of vessel elongation.We previously showed that DP1 receptor stimulation stabilizes the endothelial barrier through cAMP-PKA signaling (15).Treatment with a DP1 agonist decreased the mRNA levels of pro-angiogenic cytokines in macrophages (39).Thus, in the APB5-induced retinopathy model, H-PGDS/PGD 2 /DP1 signaling might downregulate retinal inflammation via endothelial barrier enhancement and/or macrophage cytokine expression decrease.In addition, we cannot exclude the contribution of PPARγ signaling activated by 15d-PGJ 2 .Jiang et al. reported that treatment with 15d-PGJ 2 inhibited TNFα and IL-1β expression in monocytes (40).Further investigation is required to reveal whether DP receptor signaling and/or PPARγ signaling plays a crucial role in decreasing pro-angiogenic cytokine expression during inflammation.
Here, we focused on the differences in the expression, receptive signaling, and pathophysiological activities to reveal the significance of L-PGDS and H-PGDS in the retina.Each enzyme regulates normal retinal angiogenesis depending on its expression site, activation period, and mediated receptor signaling.However, angiogenesis also plays a crucial role in several situations, such as cancer growth, rheumatoid arthritis, and tissue repair.Differences in the type of activating cells and their period complicate clarifying the mechanism of angiogenesis in each situation.Our study thus provides new insights by focusing on each enzyme in multiple models.